In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Summary

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

Abstract

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

Introduction

The optic nerve (ON), which consists of axons from the retinal ganglion cells (RGC), transmits visual signal from the retina to the brain. ON diseases, such as glaucoma, traumatic or ischemic optic neuropathy, often caused irreversible ON/RGC degeneration and devastating visual loss. Although there are currently many breakthroughs in ON regeneration and RGC protection in rodent models^1,2,3,4,5,6, clinical treatments for most of the ON diseases remaine....

Protocol

Experiments were conducted strictly in accordance with the ARRIVE guidelines and the National Institutes of Health guide for the care and use of Laboratory animals, and adhere to the protocols approved by the Institutional Animal Care and Use Committee in Wenzhou Medical University (WMU) and Joinn Laboratory (Suzhou). The male Saanen goats, aged from 4 to 6 months with weight of 19-23 kg, were housed in the WMU animal facility. The male Rhesus macaques, aged from 5 to 6 years with weight of 5-7 kg, were housed in the Joinn animal facility. All the animals were maintained in an air-conditioned room with controlled temperature (21 ± 2 °C) under a 12 h light/12....

Results

Figure 1A shows representative results of FVEP in goat. Although the waveforms in same flash intensity have relative similarity, we still recommend to examine the waveforms twice. Electromagnetic waves generated by electronic devices will interfere with the collected electrical signals, resulting in high baseline noise and poor repeatability of the waveform. Therefore, it is recommended to ensure that there are no redundant electronic devices plugged into the surrounding environment during e.......

Discussion

In this study, we present a protocol of VEP, PERG, and OCT in goat and rhesus macaque. These in vivo methods can be applied in large animal models of various optic neuropathies, such as glaucoma, ischemic, or traumatic optic neuropathy and optic neuritis⁹.

PVEP is more stable and sensitive than FVEP¹⁷; however, it can't be elicited in goat⁹. As such, FVEP is performed in goat and PVEP is performed in rhesus macaqu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
47.6 x 26.8 cm monitors	DELL Inc.	E2216HV	The visual stimuli of contrast-reversal black-white checkerboards were displayed on screens
6.0 mm tracheal tube	Henan Tuoren Medical Device Co., Ltd	PVC 6.0	ensure the airway
alligator clip
atropine	Guangdong Jieyang Longyang Animal pharmaceutical Co.,Ltd.		reduce bronchial secretion and protect heart from vagal nerve activation
Carbomer Eye Gel	Fabrik GmbH Subsidiary of Bausch & Lomb		moisten the cornea and stabilize the recording electrodes
ERG-Jet recording electrodes	Roland Consult Stasche&Finger GmbH	2300 La Chaux-De-Fonds	ERG recording
eye speculum	Shanghai Jinzhong Medical Device Co., Ltd	ZYD020	open palpebral fissure
Heidelberg Spectralis OCT system	Heidelberg Engineering		OCT system
Imaging			(https://www.heidelbergengineering.com/media/e-learning/Totara-US/files/pdf-tutorials/2238-003_Spectralis-Training-Guide.pdf)
isoflurane	RWD Life Science Co., Ltd	R510-22	isoflurane anesthesia
male Saanen goats	Caimu Livestock Company, country (Hangzhou, China)		The male Saanen goats, aged from 4 to 6 months with weight of 19–23 kg
needle electrode	Roland Consult Stasche&Finger GmbH	U51-426-G-D	use for FVEP ground electrode and PERG reference electrodes
periphery venous catheter intravenously	BD shanghai Medical Device Co., Ltd	383019	intravenous access for atropine and propofol
propofol	Xian Lipont Enterprise Union Management Co.,Ltd.		induce Isoflurane anesthesia in goat
Tropicamide Phenylephrine Eye Drops	SANTEN OY, Japan		5% tropicamide and 5% phenylephrine hydrochloride
visual electrophysiology device	Gotec Co., Ltd	GT-2008V-III	use for FVEP & PERG
xylazine	Huamu Animal Health Products Co., Ltd.		xylazine anesthesia: intramuscular injection of xylazine 3mg/kg
zoletil50	Virbac		induce Isoflurane anesthesia in monkey